Aplastic anemia (AA) and other types of bone marrow failure have clinical and laboratory features consistent with an autoimmune pathophysiology, with a diversity of putative inciting antigens, including viruses, chemicals, medical drugs, and tumor antigens. Whatever its specific etiology, a majority of patients respond with hematologic improvement after immunosuppressive therapies. One important clinical feature of AA is its association with stem cell clonal hematologic diseases, especially paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic syndromes (MDS). In the clinic, we have completed analysis of long-term follow-up of a cohort of 122 patients treated with a now standard regimen of antithymocyte globulin (ATG) and cyclosporine: survival at 7 years was highly correlated to evidence of response at 3 months, especially the robustness of improvement in platelet and reticulocyte levels. Relapse--the requirement for further immunosuppressive therapy--was frequent but usually responsive to cyclosporine or a second course of ATG. Evolution to MDS occurred in an approximate 15% of patients, usually manifest as either monosomy 7 or trisomy 8 syndrome. Accrual was terminated for a research study of 100 patients with severe AA on presentation that added mycophenolate mofetil, as well as delayed addition of cyclosporine, to standard ATG, in an effort to induce tolerance. Hematologic response rates at 3 and 6 months were comparable to those achieved by standard treatment; relapse, evolution, and survival analyses will require longer periods for confident evaluation; preliminarily, both the relapse rate and the requirement for reinstitution of cyclosporine appear lower with this regimen. Daclizumab, a monoclonal antibody that binds to the interleukin-2 receptor and has relative specificity for activated T cells, has been successfully employed in patients with moderate AA, leading to clinically significant responses in about 30% with minimal toxicity and without the need for hospitalization. Current protocols randomize previously untreated severe AA patients to either ATG plus cyclosporine for two years or to ATG, cyclosporine and rapamycin for six months. For refractory disease, patients are randomized to treatment with either rabbit ATG or CAMPATH-1, a monoclonal antibody to T cells. In the laboratory, efforts have concentrated on the incitement of AA by an unknown virus (see Z01 HL 02319-14 HB), the aberrant immune response, and the problem of clonal evolution. In efforts to more specifically characterize the immune response, we have utilized the methods of flow cytometry and spectratyping to determine expansion of V-beta TCR families of T cells and skewing of CDR3 expression within expanded families, as indicators of antigen-driven clonal T cell proliferation. For CD8 cytotoxic lymphocytes, a large proportion of AA and PNH patients show oligoclonal T cell expansion and a pattern consistent with antigen-driven T cell selection; identified individual CDR3 sequences, not present in normal blood, have identified possibly disease-specific "clonotypes" of T cells that fluctuate with disease activity and treatment response. We also have employed DNA chip analysis to examine CD34 hematopoietic cells, both normal and from patients with marrow failure syndromes. For the limited numbers of CD34 cells from AA cases target cells show increased expression of genes related to immune system activation, apoptosis, and stress, and decreased expression of proliferation genes. CD4 and CD8 T cells from AA patients show type 1 cytokine activation, and also expression of a variety of programs not previously implicated in more limited studies: genes for toll-like receptors in CD4 cells and killer-cell immunoglobulin-like receptor in CD8 cells; VCAM-1, an adhesion molecule important in other autoimmune diseases, also was upregulated. In PNH, paired primary CD34 cells from individual patients show marked upregulation of immune and apoptosis genes in the abnormal putative PIG-A-negative population; the PNH clone pattern was close to normal, and additionally there was little difference in the transciptome of CD34 cells obtained from patients with predominantly hemolytic compared to marrow failure clinical variants. In MDS, specifically up- and down-regulated genes have been observed for CD34 cells from patients with well-defined cytogenetic abnormalities in myelodysplasia . Trisomy 8, but not monosomy 7, also is associated with immune abnormalities related to hematopoiesis, as cytogenetically abnormal cells are more likely than normal cells to express Fas and to be apoptotic. We have shown that aneuploid cells in trisomy 8 are apototic, as they express Fas and annexin. The pattern of T cell usage in trisomy 8 resembles AA. Isolated CD8 T cell clones show preferential activity against cytogenetically abnormal cells, suggesting that they are reactive to a partially transformed clone of hematopoietic cells. Monosomy 7, a syndrome usually associated with a fatal course due to refractory pancytopenia or acute leukemia, chromosomally aberrrant cells are abnormally sensitive to G-CSF in vitro, and high concentrations of this cytokine appear to select for pre-existing minor populations of monosomy 7 cells only detectable by the sensitive fluorescent in situ hybridization methodology. In a mouse model of AA based on infusion of parental lymph node cells into F1 recipients, a prominent "innocent bystander" mechanism of hematopoietic stem cell killing has been demonstrated in co-transplantation experiments, likely explaining some of the extraordinary potency of limited numbers of activated T cells in human marrow failure diseases. Finally, we have implicated genes of the telomerase complex, which acts to preserve telomere length at mitosis in mammalian cells, in late onset AA and MDS. Two genese, TERC (for the RNA component) and DKC1, cause the constitutional AA dyskeratosis congenita. In studies of kindreds with probands presenting with AA, without physical stigmata but with other family members showing mild hematologic abnormalities, we identified two novel mutations in TERC in all affected members. Despite only mild anemia, erythrocyte macrocytosis, or thromboycotopenia, marrows were strikingly hypocellular and showed low content of CD34 cells and functional colony-forming progenitor cells. One family member had been misdiagnosed with MDS at an advanced age, and both he and the proband showed good therapeutic response to instition of androgens (frequently effective in inherited marrow failure syndromes). Most recently, we have discovered mutations in the TERT gene for the telomerase enzyme itself, not previously associated with disease in humans, in four patients with onset of marrow failure in middle-age. Histocompatability antigens and cytokine promoter polymorphisms have been suggested as immune system risk factors for AA; we now propose TERC and TERT abnormalities as responsible, through acclerated telomere shortening, for greatly diminished stem cell compartments and therefore hematopoietic system risk factors for marrow failure.